Haematococcus is a group of green microalgae which are able to accumulate a large amount of a keto-carotenoid, astaxanthin, under certain environmental conditions (Boussiba et aL 1992). Astaxanthin is the major pigment imparting the red-pinkish color of salmon, trout and shrimp, whose prices are largely determined by their color. Currently, the major commercially available source of astaxanthin is in the form of a synthetic product, which is expensive (&gt;US$ 3,000 per kilogram) and may contain astaxanthin compounds having an unnatural configuration. There is now a trend toward using natural sources of feed nutrients for the purpose of preparing feed for the above types of fish, and thus a less expensive and natural source of astaxanthin has been sought. This group of green microalgae appears to be the most promising source for this purpose. Although astaxanthin can be synthesized by other algae, bacteria and a few fungi (Schroeder & Johnson 1993; Yokoyama & Miki 1995), the amounts accumulated by Haematococcus (Boussiba & Vonshak 1991), however, surpass those obtained in the above noted sources. According to the present development of aquaculture, it is expected that by the year 2000, more than 100 tons of astaxanthin will be required (Johnson & An, 1991; Borowitzka, 1992). This potential demand could open a large market for microbially produced astaxanthin. Several reasearch groups have attempted to establish large production systems for Haematococcus.
However, no one has succeeded so far, due to the difficulties in enhancing astaxathin accumulation rate, preventing contamin ation by other microorganisms, and failure in special photobioreator design. An example of previous methods for producing astaxanthin and other pigments from Haematococcus is that described in the international patent application No. WO 89/06910.
H. pluvialis has a unique life cycle comprising two stages: a green, motile, vegetative stage in which the cells continuously divide and synthesize chlorophyll and a red, non-motile resting stage (cyst) in which cell division stops, chlorophyll content remains constant and astaxanthin content and cellular dry weight continuously increase. These two stages are illustrated in FIG. 1, which depicts graphically the relationship between four parameters of the growth of H. pluvialis, namely, chlorophyll, astaxanthin, dry weight and cell number. The optimal environ mental and nutritional conditions required for these two stages are quite different. For vegetative growth, full nutrient medium, moderate light intensity and adequate temperature and pH are essential. For the resting stage, however, no nutrient (except carbon) is required and higher light intensity (e.g., sunlight) is necessary for faster astaxanthin accumulation. Due to such discrepancies, the two stages must be separated into different cultivating systems with different medium accordingly. The growth strategies for each stage are also different. In the green stage, the optimal conditions for cell division are maintained to achieve the ma um cell number. In the red stage, the optimal inductive conditions must be provided for astaxathin accumulation. Heretofore, no such process has been developed for the effective large scale, two-stage or two-phase growth of Haematococcus cells, which answers the demands of each of the above growth stages of Haematococcus cells. The known processes suffer from a number of drawbacks, including the need for expensive growth media, apparatuses, use of sterile equipment and media and/or the use of expensive antibiotics or other anti-microbial agents to prevent contamination of the desired Haematococcus cultures by other microorganisms such as other microalgae, fungi, yeasts and bacteria. Thus, such processes are expensive and time-consuming to perform, and they also do not always provide a high yield of Haematococcus cells and/or of astaxathin in the cultivated cells.
It is therefore one aim of the invention to provide a new two-phase cultivating procedure which allows for the production of vegetative green Haematococcus cells under controlled conditions of temperature, light intensity and nutrients and for the induction and production of red Haematococcus cells rich in astaxnthin under less stringently controlled conditions, of temperature and light, but under conditions of nutrient stress which accelerates the induction and accumulation of astaxanthin in the cells.
Likewise, it is another aim of the present invention to provide a process for the large-scale production of astaxanthin-enriched Haematococcus cells by utilization of the above-mentioned two-phase cultivating procedure.
Other aims of the present invention will be readily apparent from the following disclosure of the invention.